By investigating the lifetime of the excited states of a sample or a fluorophore, important conclusions can be drawn as to the properties of the sample. Especially when multiple fluorescent dyes are used, identification and distinction of the fluorophores becomes possible using fluorescence lifetime image microscopy (FLIM).
EP 0 681 695 B1 discloses an apparatus for quantitative imaging of multiple fluorophores. The apparatus contains a means for guiding two scanning beams of continuous electromagnetic radiation of different wavelengths. The intensity of each beam is sinusoidally modulated with various modulation frequencies. The modulated detected radiation is allocated to the respective excitation wavelengths using the lock-in technique.
DE 101 44 435 A1 discloses a method and an arrangement for generating time-resolved and positionally resolved, as well as time- and wavelength-resolved, fluorescence images. The arrangement is based on pulsed fluorescence excitation with a femtosecond or picosecond laser, detection being accomplished in time-correlated and positionally correlated fashion in a single-photon counting system.
In scanning microscopy, a sample is illuminated with a light beam in order to observe the reflected or fluorescent light emitted from the sample. The focus of the illuminating light beam is moved in a specimen plane by means of a controllable beam deflection device, generally by tilting two mirrors, the deflection axes usually being perpendicular to one another so that one mirror deflects in the X direction and the other in the Y direction. Tilting of the mirrors is brought about, for example, by means of galvanometer positioning elements. The power level of the light coming from the specimen is measured as a function of the position of the scanning beam.
In confocal scanning microscopy specifically, a specimen is scanned in three dimensions with the focus of a light beam.
A confocal scanning microscope generally comprises a light source, a focusing optical system with which the light of the source is focused onto an aperture (called the “excitation pinhole”), a beam splitter, a beam deflection device for beam control, a microscope optical system, a detection pinhole, and the detectors for detecting the detected or fluorescent light. The illuminating light is coupled in via a beam splitter. The fluorescent or reflected light coming from the specimen travels back via the beam deflection device to the beam splitter, traverses it, and is then focused onto the detection pinhole behind which the detectors are located. Detected light that does not derive directly from the focus region takes a different light path and does not pass through the detection pinhole, so that a point datum is obtained that results, by sequential scanning of the specimen, in a three-dimensional image. A three-dimensional image is usually achieved by acquiring image data in layers.
In confocal scanning microscopy, a detection pinhole can be dispensed with in the case of two-photon (or multi-photon) excitation, since the excitation probability depends on the square of the photon density and thus on the square of the illuminating light intensity, which of course is much greater at the focus than in the adjacent regions. The fluorescent light being detected therefore very probably originates almost exclusively from the focus region, which renders superfluous any further differentiation, using a pinhole arrangement, between fluorescent photons from the focus region and fluorescent photons from the adjacent regions.
In this case non-descan detection can be performed, in which the detected light does not (as in the case of the descan configuration) travel to the detector via the beam deflection device and through the beam splitter for incoupling illuminating light, but instead is deflected out directly after the objective by means of a dichroic beam splitter, and detected. Arrangements of this kind are known, for example, from the publication of David W. Piston et al., “Two-photon excitation fluorescence imaging of three-dimensional calcium ion activity,” Applied Optics, Vol. 33, No. 4, February 1996, and from Piston et al., “Time-Resolved Fluorescence Imaging and Background Rejection by Two-Photon Excitation in Laser Scanning Microscopy,” SPIE Vol. 1640.
Lifetime imaging based on time-correlated single-photon measurement is usually implemented using infrared pulsed lasers with a repetition rate of approx. 80 MHz, with multi-photon (usually two-photon) excitation. These pulsed infrared lasers (usually mode-coupled titanium sapphire lasers) are very expensive and very complex. The pulse repetition rate of these lasers depends directly on the resonator length and therefore cannot be varied. With longer-lifetime fluorescent dyes in particular, there is a greater probability that excited fluorophores will not have returned to the ground state by the time the next excitation pulse arrives. Because the next excitation pulse serves as the time base in such cases, this results in incorrect measurement results. A further disadvantage of the known arrangements based on pulsed titanium sapphire lasers derives from the fact that because of their design, titanium sapphire lasers emit excitation light in the region of approximately 800 nm (approx. 720-980 nm), so that only dyes matched specifically to those wavelengths can be investigated and used.